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Il ruolo del PTP in apoptosi/necrosi

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Presentation on theme: "Il ruolo del PTP in apoptosi/necrosi"— Presentation transcript:

1 Il ruolo del PTP in apoptosi/necrosi

2 Disease Some characteristics common to several diseases:
Oxidative damage Changes in Calcium homeostasis Loss of energy production Cell death Tissue and organ dysfunction Disease

3 The Mitochondrial connection.
Mitochondria are key integrators of cellular signals and stress: make life and death decisions IMM c OMM c c c c c Bax/Bcl2 mPTP Swelling, rupture Release of proapoptotic factors Apoptosis and necrosis are facilitated at the level of the mitochondria by the opening of large pores (excluding extrinsic apoptotic pathway)

4 The Mitochondrial Permeability Transition.
Mitochondrial Permeability transition (mPT) characterized by: Loss of inner membrane potential; Release of Cytochrome C; Cell death by apoptosis or necrosis; Cessation of mitochondrial respiration; Release of mitochondrial Ca 2+; Mitochondrial swelling (rupture) The mPT can be caused by several agents/mechanisms (e.g. ROS, Ca2+ overload); The mPT, and as a consequence, mitochondrial dysfunction, is associated with the onset and progression of several diseases. ANT: Adenine nucleotide transporter, VDAC: voltage dependent anion channel, HK: Hexokinase; CyD: Cyclophilin D, CK: Creatine kinase, PBR: Periferal benzodiazepin receptor

5 Recenti sviluppi nella identificazione delle componenti del PTP
La ATP sintetasi, maggiore produttore cellulare di ATP nella cellula, è stata recentemente proposta come l’unico componente molecolare necessario per la formazione del PTP: questo dato è ancora da confermare in maniera definitiva, ma enfatizza la funzione duale dei componenti della catena respiratoria nel controllo di apoptosi e necrosi (che ritroveremo con il citocromo C)

6 Consequences of mPTP opening
stimulus mPTP opening solutes across mPTP enter mitochondria loss of membrane potential/energy production mitochondria swell and outer membrane ruptures mitochondria swell and outer membrane rupture Ca++ and protein release (eg. Cytochrome c) Cell death

7 The mPTP and Disease. Opening of mPTP has been implicated in the aetiology and progression of several diseases including : Neurodegenerative diseases (Parkinsons, Alzheimers, MS, Huntingtons, ALS) Ischemia / reperfusion injury (AMI, Stroke, organ transplantation) Dystrophies (Bethlem, Ullrich, Duchene) Diabetic Complications (retinopathy, nephropathy) Oxidative damage, irregular calcium signalling and mitochondrial dysfunction is common to all of these diseases

8 Take a closer look at Ischemia / reperfusion injury
(from a Mitochondrial standpoint only)

9 Lethal reperfusion Injury.
Heart ischemia leads to infarct size of > 70%. Reperfusion of tissue can reduce infarct size. Paradoxically reperfusion also leads to tissue death and contributes to infarct size (LRI). Methods to reduce lethal reperfusion injury should have clinical benefit

10 PTP in Ischemia / Reperfusion Injury
What’s happening in the the cell and mitochondria: Ca2+ Calcium enters mitochondria via the Calcium Uniporter (not saturated at High [Ca2+]) Ca2+ Uniporter Normal conditions Calcium is pumped out of the mitochondria via the Sodium / Calcium Antiporter (saturated at High [Ca2+]) Na/Ca2+ Antiporter Mitochondria Cell Under conditions of high cytoplasmic [Ca2+] the mitochondria can overload with calcium and this can cause the mPT.

11 PTP in Ischemia / Reperfusion Injury
Cell Restart to respiration. Large burst of ROS production. Mitochondria load with calcium via uniporter and saturate the Na+/Ca2+ antiporter. Reoxygenation conditions Ca2+ Ca2+ Uniporter Na/Ca2+ Antiporter Mitochondria Mitochondria Cell Calcium overload and oxidative damage from ROS production induce opening of the mPTP and mitochondrial dysfunction. Cell death via necrosis/apoptosis depending on damage. Reoxygenation conditions mPTP Ca2+ CytC Cell Death Necrosis Apoptosis Mitochondria Cell High intracellular [Na+] causes reversal of the Na+/Ca2+ Antiporter and the cell loads with Ca2+. Na+ Na+/Ca2+ Antiporter Ischemic conditions Ca2+ Mitochondria Cell To counter the high [H+] the cell uses the Na+/H+ Antiporter. Due to lack of ATP the Na+ can not be pumped out and the cell loads with Sodium. Na+/H+ Antiporter Na+ H+ Ischemic conditions Ischemic conditions Mitochondria Cell Loss of ATP production via respiration; increase in lactic acid and a drop in cellular pH. pH [H+] H+

12 Analisi di topi KO per la ciclofilina D

13 I topi KO hanno un difetto nell’apertura del PTP, ma sono normali
Apertura del PTP in mitocondri isolati

14 Cellule PTP -/- sono resistenti alla morte cellulare indotta da ROS (H2O2)

15 I topi KO sono resistenti al danno cardiaco indotto da ischemia-riperfusione

16 From Yellon and Hausenloy (2007 NEJM)
New cardioprotective strategies. mPTP inhibitors could be a promising strategy for lethal reperfusion injury. From Yellon and Hausenloy (2007 NEJM)

17 Inibitori del PTP (CsA) sono anche efficaci nella riduzione del danno post-ischemico dell’Uomo
Marker sierico Area infarto (NMR)

18 The mPTP is involved in the pathogenesis of
several aging-associated diseases Studies using CsA and Ppif-/- mice (cyD null) have highlighted a potential role for the mPTP in the progression of several diseases. Reperfusion Injury Neurodegenerative Disease Dystrophies Myocardial infarction Alzheimer’s Disease ALS Duchenne MD Piot et al 2008 Millay et al, 2008 Du et al, 2009 Keep et al, 2001 Stroke Ullrich / Belthem Ppif -/- mice and CsA Korde et al, 2007 Palma et al, 2009 Other Coronary Artery Bypass Graft Heart Disease Transplantation and Surgery Diabetic Retinopathy Hyperglycemia Doxorubicin toxicity Platelet activation Traumatic brain injury Non-alcoholic fatty liver disease

19 L’identificazione di nuovi farmaci inibitori dell’apertura del PTP

20 A large network of industry/academia interactions
Hamburg Oxford: EVOTEC Lyon: M Ovize Milan: CONGENIA PG Pelicci R Latini Nikem NMS Padua: P Bernardi F Di Lisa

21 Innovative HTS unprecedented at this scale Organelle based
Requiring the use of animals->liver mitochondria unprecedented at this scale A high pulse of Ca2+ (ca. 150 mM) given to purified mitochondria will cause mPTP opening and induce mitochondrial swelling (DA540nm). A Ca2+-induced swelling assay was developed suitable for HTS Fresh Rat liver mitochondria were prepared daily and used in this assay to screen >300,000 compounds Counterscreens were run to elimate compounds that interfere with mitochondrial respiration. Several chemical classes were identified Ca μM 1 mM CsA Control Absorbance (A 540 nm) Time (mins) 5 10

22 Adaptation of mitochondrial assays to 1536 well format
Parameters to optimize: density / fitness of mitochondria concentration of stimulus (Ca++) assay sensitivity (CysA) reagents volumes / order of addition reagents incubation times reagents dispensing devices readout kinetics / stability of assay signal DMSO-sensitivity reader settings (i.e. OD filter sets) Results of time-course experiment in 1536 well plates: 4 Fitzones Z‘: 0,74 / 0,76 / 0,70 / 0,61 Mitochondria: 1 mg/ml Calcium: 300 µM Incubation: 10 min Cyclosporin A: 1 µM

23 HTS-Hardware in Operations
2 EVOscreen® Mark II: biochemical assays at 1 µL level 1 EVOscreen® Mark III: biochemical & cellular assays at 1-10 µL level

24 PTPi block mPTP opening by several stimuli
Absorbance (A 540 nm) Time (mins) Ca2+ 150μM, pH 7.4 Treated GNX-A Untreated Class 6 compounds inhibit the mPT induced by: Calcium overload Oxidative damage Protein crosslinkers Respiration uncouplers GNX-A GNX-A GNX-A Untreated Untreated A 540 nm Untreated A 540 nm A 540 nm Treated Treated Treated Time (mins) Time (mins) Time (mins) 50nM FCCP 40μM Ca2+, pH 7.4, 100μM Menadione 40μM Ca2+, pH 7.4, 300μM Diamide, 40μM Ca2+, pH 7.2, Assay performed on prepared mouse hepatic mitochondria

25 PTPi do not have significant effects on mitochondrial respiration
State 3 inhibition (with ADP) State 3 inhibition (with ADP) -30 -20 -10 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 21 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 5 10 15 20 25 % max inhibition % max inhibition [GNX-C] (mM) [GNX-B] (mM) State 4 inhibition State 4 inhibition 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 -20 -10 30 40 50 60 70 80 90 100 1 2 -10.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 % max inhibition % max inhibition [GNX-C] (mM) [GNX-B] (mM)

26 The identified PTP inhibitors are >> than CsA
Class 6 compounds are able to increase the CRC (by mPTP inhibition) several fold over that of unprotected mitochondria. Class 6 compounds increase the CRC significantly over that maximally attainable with CsA. Control CsA (1μM) GNX-B (0.1mM) Ca2+ Pulses (10 mM) Extra-mitochondrial Calcium fluorescence Ca2+ Release mPTP open GNX-B (1.0mM) GNX-B (5.0mM) 40 mM 120 mM 250 mM 180 mM 380 mM Time (min) CRC (mM Ca2+) GNX C 0.1 mM 0.5 1.0 5.0 50 100 150 200 250 300 350 CsA Control The Calcium Retention Capacity (CRC) of purified mouse liver mitochondria is determined by measuring the point at which pulse-loaded calcium is released from the mitochondria. After the addition of a pulse of calcium (10 mM) the extramitochondrial fluorescence increases. As the mitochondria take-up the calcium the fluorescence signal decreases. Calcium is continually loaded into the mitochondria until there is a sudden, large, increase in calcium fluorescence which indicates complete release of the stored calcium due to opening of the mPTP. Inhibition of the mPTP, with Cyclosporin A or our proprietary inhibitors can increase the capacity of mitochondria to retain calcium and thus protect the mitochondria from calcium overload in stress situations. Extramitochondrial calcium is measured by the fluorescence of calcium green. Note, increasing the concentration of CsA does not increase the CRC due to saturation of its target (Cyclophilin D) and is one of its limitations.

27 PTP inhibitors for Acute Myocardial Infarction
The highway to proof-of-concept: unmet medical need strong evidence for involvment of the mPTP linear path for the design of a clinical study

28 PTP inhibitors are cardioprotective
in animal models Area at Risk (g) Arae Necrotic (g) vehicle A-B GNX -B CsA 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1.0 1.2 1.4 No protection Protection . New Zealand White Rabbits were subjected to Left Anterior Descending (LAD) coronary artery occlusion for 30 mins followed by 4 hrs of reperfusion. Area at Risk and infarcted area (area necrotic) was determined by Evans blue and TTC staining. GNX-B (15 mg /kg in 3 mls 40% PEG 400; 20% DMSO) and CsA (10 mg/kg in sandimune) were administered by i.v. bolus 5 mins prior to reperfusion. Note, as with CsA in the clinical proof of concept trial, there is greater protection when the area at risk is larger. (In collaboration with Prof Ovize, Ospice di Lyon)

29 Un ruolo inaspettato per p53 nell’induzione di necrosi
Dopo stress, p53 si trova localizzata insieme con la ciclofilina-D ai mitocondri

30 P53 è richiesta per l’apertura del PTP durante la necrosi da stress ossidativo
Viene misurata l’apertura del PTP

31 Lo stress ossidativo induce necrosi e non apoptosi che dipende da p53

32 Considerazioni P53 è un regolatore fondamentale dell’apoptosi, ma sembra giocare un ruolo altrettanto rilevante nella morte cellulare da necrosi dopo stress ossidativo P53 è “good” e “bad”: in questo caso potrebbe essere coinvolto nella patogenesi di molte malattie Il coinvolgimento di p53 è un’altra dimostrazione che anche la necrosi è sottoposta (almeno in alcuni casi) a un livello di regolazione impensabile fino a poco tempo fa

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